Abstract Chronic wounds impact ~6.5M people and cost ~$25B per year in the US alone. Despite significant effort, understanding the mechanisms involved in development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in humans and because current animal models are inadequate. The PI and team have developed a novel mouse model for diabetic chronic wounds that closely mimics those of humans. Low levels of oxidative stress (OS) are important for proper healing; however, when OS levels are high wound healing does not occur. Human chronic wounds have high levels of OS, but it is not known if OS is critical for developing chronicity. Using diabetic mice, we can generate chronic wounds 100% of the time by creating high levels of OS immediately after wounding by treating with inhibitors specific to two antioxidant enzymes. The wounds become fully chronic within 20 days after treatment and remain chronic until the mouse dies, sometimes >100 days. The wounds in the mouse model feature all of the same problems observed in human chronic diabetic wounds: high levels of OS lead to DNA damage, gene deregulation, protein and lipid damage, cell death, impaired keratinocyte migration (potentially inhibiting re- epithelialization), chronic inflammation, and lack of proper angiogenesis and deposition of matrix resulting in poor development of the healing new tissue. Equally important, the chronic wounds in the mouse model develop a biofilm from the bacteria present on the skin by elimination of non-biofilm-forming bacteria in favor of the biofilm-forming species. These biofilm-forming bacteria are also present on human skin and appear in human diabetic chronic wounds. All of these characteristics indicate that the PI's mouse model mimics all key aspects of human chronic wounds. The PI hypothesizes that high levels of OS induce changes in the dynamics of the commensal microbiome resulting in a transition to a biofilm-forming microbiome and development of the biofilm. This hypothesis will be investigated through the following studies: Aim #1: Identify the composition of the microbiome to characterize biofilm initiation and progression and develop a longitudinal statistical classifier that enables prediction of biofilm-forming bacteria in chronic wounds. Aim #2: Determine whether OS is necessary and sufficient for biofilm development. This project will use a novel model of chronic wounds developed in the PI's laboratory that has all the tissue characteristics present in human chronic wounds, contains the major bacterial pathogens present in human chronic wounds and develops a biofilm naturally, i.e. without the need to introduce bacteria from external sources. Therefore, this model will allow, for the first time, studies of the natural progression and dynamics of biofilm development in vivo. The results from these exploratory studies will serve as the basis for future mechanistic studies to determine how OS induces biofilm development. These studies will impact the field by providing, for the first time, a better understanding of whether specific bacteria can be used as prognostic tools for development of wound chronicity in mice. This work will provide insight into whether the microbiome can be exploited as a marker for healing/non-healing after debridement and in the future help with the design of proof-of-concept experiments using human specimens.